The replication cycle of a typical eukaryotic somatic cell consists of four phases: G.sub.1, S (DNA synthesis), G.sub.2, and M (mitosis) The result of this process is the generation of two daughter cells that are equivalent both in genetic makeup and in size to the original parental cell. Feedback controls operating at checkpoints ensure the faithful replication and segregation of the genetic material. In eukaryotic organisms, a general paradigm has emerged in which a family of proteins, called cyclins, and cyclin-dependent protein kinases (Cdks) regulate cell cycle progression. These mechanisms are at the level of reversible phosphorylation, binding to low-molecular-weight inhibitors, transcription, intracellular compartmentalization, and protein degradation.
The transition from G.sub.2 to M phase requires the activity of M-phase-promoting factor (MPF), which is composed of Cdc2, an evolutionarily conserved serine/threonine-specific protein kinase, and B-type cyclins. The activity of Cdc2 is regulated not only by is association with B-type cyclins but also by reversible phosphorylation. Proper regulation of MPF ensures that mitosis occurs only after earlier phases of the cell cycle have been completed successfully. This strict control of MPF is largely post-translational, involving the phosphorylation of Cdc2 at three key residues. After Cdc2 associates with cyclin, the cyclin-dependent kinase (CDK)-activating kinase (CAK) phosphorylates Cdc2 on Thr.sup.161. This phosphorylation would generate active MPF, but two additional phosphorylations on Thr.sup.14 and Tyr.sup.15 of Cdc2 suppress MPF activity during interphase. At the G.sub.2 -M transition, the Cdc25 protein dephosphorylates Thr.sup.14 and Tyr.sup.15, thereby allowing MPF to phosphorylate its mitotic substrates.
Phosphorylation on Thr.sup.14 and Tyr.sup.15 maintains Cdc2 in an inactive state throughout the S and G.sub.2 phases of the cell cycle, and Thr.sup.161 phosphorylation is required for the kinase activity of the complex. Dephosphorylation of both Thr.sup.14 and Tyr.sup.15 by the Cdc25 phosphatase in late G.sub.2 activates Cdc2 and is an obligate step for the onset of mitosis. Exit from mitosis requires the proteolytic degradation of the B-type cyclins, which is mediated by ubiquitination.
Various genetic and biochemical studies have indicated that Wee1 is the kinase that phosphorylates Cdc2 on Tyr.sup.15. Wee1 was originally identified in the fission yeast Schizosaccharomyces pombe as a critical negative regulator of mitosis. Subsequently, a second S. pombe homolog (Mik1) and Wee1 homologs from at least six other organisms have been found. In human and Xenopus, Wee1 is a soluble enzyme that phosphorylates Cdc2 on Tyr.sup.15, but not on Thr.sup.14.